Ginsenoside Re inhibits pacemaker potentials via adenosine triphosphate-sensitive potassium channels and the cyclic guanosine monophosphate/nitric oxide-dependent pathway in cultured interstitial cells of Cajal from mouse small intestine

Abstract

Background: Ginseng belongs to the genus Panax. Its main active ingredients are the ginsenosides. Interstitial cells of Cajal (ICCs) are the pacemaker cells of the gastrointestinal (GI) tract. To understand the effects of ginsenoside Re (GRe) on GI motility, the authors investigated its effects on the pacemaker activity of ICCs of the murine small intestine.

Methods: Interstitial cells of Cajal were dissociated from mouse small intestines by enzymatic digestion. The whole-cell patch clamp configuration was used to record pacemaker potentials in cultured ICCs. Changes in cyclic guanosine monophosphate (cGMP) content induced by GRe were investigated.

Results: Ginsenoside Re (20-40μM) decreased the amplitude and frequency of ICC pacemaker activity in a concentration-dependent manner. This action was blocked by guanosine 5'-[β-thio]diphosphate [a guanosine-5'-triphosphate (GTP)-binding protein inhibitor] and by glibenclamide [an adenosine triphosphate (ATP)-sensitive K(+) channel blocker]. To study the GRe-induced signaling pathway in ICCs, the effects of 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (a guanylate cyclase inhibitor) and RP-8-CPT-cGMPS (a protein kinase G inhibitor) were examined. Both inhibitors blocked the inhibitory effect of GRe on ICC pacemaker activity. L-NG-nitroarginine methyl ester (100μM), which is a nonselective nitric oxide synthase (NOS) inhibitor, blocked the effects of GRe on ICC pacemaker activity and GRe-stimulated cGMP production in ICCs.

Conclusion: In cultured murine ICCs, GRe inhibits the pacemaker activity of ICCs via the ATP-sensitive potassium (K(+)) channel and the cGMP/NO-dependent pathway. Ginsenoside Re may be a basis for developing novel spasmolytic agents to prevent or alleviate GI motility dysfunction.

Keywords: gastrointestinal tract; ginsenoside Re; interstitial cells of Cajal; patch clamp configuration.

Fig. 1

Cultured ICCs from the murine small intestine. The tunica muscularis of the small bowel was digested with collagenase, and the dispersed cells were cultured for 12 h. The confocal microscope image shows the Kit-immunopositive ICC network in the culture. The scale bar represents 50 μm. GRe, ginsenoside Re; ICC, interstitial cells of Cajal.

Fig. 2

The effect of GRe on the pacemaker activity of cultured ICC clusters. (A–D) The pacemaker activity of interstitial cells of Cajal (ICCs) exposed to GRe (20–40μM) in the current-clamp mode (I = 0). Ginsenoside Re decreased the amplitude and frequency of the ICC pacemaker activity in a concentration-dependent manner. (E,F) The graphs summarize the responses to GRe. The bars represent the mean ± the standard deviation. ** p < 0.01. CTRL, control; GRe, ginsenoside Re; ICC, interstitial cells of Cajal.

Fig. 3

The effects of TEA, 4-aminopyridine, apamin, and glibenclamide on pacemaker activity inhibition by GRe in cultured ICC clusters. Pretreatment with (A) TEA (10mM), (B) 4-aminopyridine (5mM), or (C) apamin (1μM) did not affect the inhibitory effects of GRe. (D) Pretreatment with glibenclamide (10μM) blocked the inhibitory effects of GRe. (E) The GRe-induced inhibitory effects were reversed by adding glibenclamide. (F) The graph summarizes the responses to GRe in the presence of potassium channel blockers. The bars represent the mean ± the standard deviation. ** p < 0.01. 4-ami., 4-aminopyridine; CTRL, control; Gliben., glibenclamide; TEA, tetraethylammonium chloride.

Fig. 4

The effects of GDPβS in the pipette on pacemaker activity inhibition by GRe in cultured ICC clusters. (A) The pacemaker activity of ICCs exposed to GRe in the presence of GDPβS (1mM) in the pipette solution. Under these conditions, the inhibitory effects of GRe on pacemaker activity were blocked. (B) The graph summarizes the responses to GRe in the presence of GDPβS in the pipette. The bars represent the mean ± the standard deviation. ** p < 0.01. CTRL, control; GDPβS, guanosine 5′-[β-thio]diphosphate; GRe, ginsenoside Re; ICC, interstitial cells of Cajal.

Fig. 5

The effects of SQ-22536 (an adenylate cyclase inhibitor), ODQ (a guanylate cyclase inhibitor), RP-8-CPT-cGMPS (a PKG inhibitor) and L-NAME (a nonselective nitric oxide synthase inhibitor) on pacemaker activity inhibition by GRe in cultured ICC clusters. (A) The pacemaker activity of ICCs exposed to GRe in the presence of SQ-22536 (100μM). SQ-22536 had no effect on the inhibition of pacemaker activity by GRe. The pacemaker activity of ICCs exposed to quercetin in the presence of (B) ODQ (100μM), (C) RP-8-CPT-cGMPS (10μM), or (D) L-NAME (100μM). The inhibitors ODQ, RP-8-CPT-cGMPS, and L-NAME blocked pacemaker activity inhibition by GRe. (E) The graph summarizes the response to GRe in the presence of SQ-22536, ODQ, RP-8-CPT-cGMPS, and L-NAME. The bars represent the mean ± the standard deviation. ** p < 0.01. CTRL, control; GRe, ginsenoside Re; ICC, interstitial cell of Cajal; L-NAME, L-NG-nitroarginine methyl ester; ODQ, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; PKG, protein kinase C; RP, RP-8-CPT-cGMPS.

Fig. 6

The effect of cGMP production on pacemaker activity inhibition by GRe in cultured ICC clusters. Preincubation of the ICCs with GRe significantly stimulated cGMP production. The bars represent the mean ± the standard deviation. * p < 0.05. cGMP, cyclic guanosine monophosphate; CTRL, control; GRe, ginsenoside Re; ICC, interstitial cell of Cajal.